This study will investigate proteasome inhibition as a novel mechanism to augment the expression of nonviral gene delivery systems in vivo. The gene delivery system under investigation is a multi-component peptide condensed targeted gene delivery system. The key components consist of plasmid DNA condensed by a sulfhydryl cross-linking polymer composed of polyethylene glycol (PEG)-peptide, targeting glycopeptide, and fusogenic peptide. Specific targeting is achieved using glycopeptides that bind to either the asialoglycoprotein receptor on hepatocytes or the mannose receptor on Kupffer cells. Following internalization via receptor mediated endocytosis, sulfhydryl cross-linked DNA condensates depolymerize in the reducing environment of the endosome. Released fusogenic peptides facilitate endosomal lysis and DNA escape into the cytosol. Following condensate un-coating, DNA targets the nucleus. The efficiency of gene transfer is limited by the ability of DNA condensates to escape endosomes and avoid degradation in the cytosol. Preliminary studies indicate the proteasome metabolizes peptide-DNA condensates in the cytosol, resulting in premature degradation of plasmid DNA by cytosolic DNAse. The present proposal will investigate the use of proteasome inhibitors to block DNA condensate metabolism in the cytosol and enhance gene transfer efficiency. The central hypothesis to be tested is that proteasome inhibition will enhance the level and duration of transient gene expression in vivo by stabilizing plasmid DNA from metabolism. The proposed studies aim to incorporate intrinsic peptide-based proteasome inhibitors into sulfhydryl cross-linked non-viral gene delivery systems that target either hepatocytes or Kupffer cells. The overall objective of the proposed study is to increase the efficiency of non-viral gene delivery using a novel mechanism of proteasome inhibition to block DNA condensate metabolism. The successful outcome of these studies should provide a rational approach to develop more efficient gene delivery systems that can be used to treat a variety of human diseases.